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Abstract:

A method for preserving viral particles comprises: (i) providing an
aqueous solution of one or more sugars, a polyethyleneimine and said
viral particles wherein the concentration of polyethyleneimine is 15
μM or less based on the number-average molar mass (Mn) of the
polyethyleneimine and the sugar concentration or, if more than one sugar
is present, total sugar concentration is greater than 0.1 M; and (ii)
drying the solution to form an amorphous solid matrix comprising said
viral particles.

Claims:

1. A method for preserving viral particles comprising:(i) providing an
aqueous solution of one or more sugars, a polyethyleneimine and said
viral particles wherein the concentration of polyethyleneimine is less
than 500 nM based on the number-average molar mass (Mn) of the
polyethyleneimine and the sugar concentration or, if more than one sugar
is present, total sugar concentration is greater than 0.1M; and(ii)
drying the solution to form an amorphous solid matrix comprising said
viral particles.

2. The method according to claim 1 wherein the concentration of
polyethyleneimine is between 0.0025 and 200 nM, based on the
number-average molar mass (Mn) of the polyethyleneimine.

3. The method according to claim 1 wherein the concentration of
polyethyleneimine is between 0.025 and 200 nM, based on the
number-average molar mass (Mn) of the polyethyleneimine.

4. The method according to claim 1 wherein the concentration of
polyethyleneimine is less than 100 mM based on the number-average molar
mass (Mn) of the polyethyleneimine.

5. The method according to claim 1 in which the Mn of the
polyethyleneimine is between 20 and 1000 kDa and the concentration of the
polyethyleneimine is between 0.001 and 100 nM based on the Mn.

6. The method according to claim 1 wherein the Mn of the
polyethyleneimine is between 300 and 2000 Da.

7. The method according to claim 1 in which the sugar concentration, or
total sugar concentration, is between 0.5 and 2M.

8. The method according to claim 1 in which the sugar is sucrose,
stachyose, raffinose or a sugar alcohol.

9. The method according to claim 1 wherein a solution of two or more
sugars is used.

10. The method according to claim 9 wherein the concentration of sucrose
relative to the other sugar is at a ratio of between 3:7 and 9:1; and the
concentration of polytheyleneimine based on Mn in step (i) is
between 0.0025 nM and 100 nM.

11. The method according to claim 1 in which the admixture is
freeze-dried.

12. The method according to claim 1 in which the viral particles are
composed of a live virus or killed virus.

13. The method according to claim 12 in which the live virus is whole
virus or live-attenuated virus.

14. An excipient for the preservation of viral particles comprising:(a)
sucrose, stachyose or raffinose or any combination thereof; and(b)
polyethylenimine at a concentration based on Mn of less than 500 nM.

15. (canceled)

16. A kit comprising the excipient according to claim 14.

17. A method of preparing a vaccine comprising viral particles, which
method comprises:(a) providing an aqueous solution of one or more sugars,
a polyethyleneimine and said viral particles wherein the concentration of
polyethyleneimine is less than 500 nM based on the number-average molar
mass (Mn) of the polyethyleneimine and the sugar concentration or,
if more than one sugar is present, total sugar concentration is greater
than 0.1M; and(b) optionally adding an adjuvant, buffer, antibiotic
and/or additive to the admixture; and(c) drying the solution to form an
amorphous solid matrix comprising said viral particles.

18. The method according to claim 17 in which the vaccine is a multivalent
vaccine.

[0002]Some biological molecules are sufficiently stable that they can be
isolated, purified and then stored in solution at room temperature.
However, this is not possible for many materials and techniques involving
storage at low temperature, addition of stabilisers, freeze-drying,
vacuum formation and air-drying have been tried to ensure shelf
preservation. Despite the availability of these techniques, some
biological materials still show unsatisfactory levels of stability during
storage and some techniques lead to added cost and inconvenience. For
example, refrigerated transportation and storage is expensive. Further,
refrigerated transport is often not available for the transport of
medicines such as vaccines in countries in the developing world.

[0003]In particular, the stresses of freeze-drying or lyophilisation can
be very damaging to some biological materials. Freeze drying of
biopharmaceuticals involves freezing solutions or suspensions of
thermosensitive biomaterials, followed by primary and secondary drying.
The technique is based on sublimation of water at subzero temperature
under vacuum without the solution melting. Freeze-drying represents a key
step for manufacturing solid protein and vaccine pharmaceuticals. The
rate of water vapour diffusion from the frozen biomaterial is very low
and therefore the process is time-consuming. Additionally, both the
freezing and drying stages introduce stresses that are capable of
unfolding or denaturing proteins.

[0004]WO-A-2006/0850082 reports a desiccated or preserved product
comprising a sugar, a charged material such as a histone protein and a
dessication- or thermo-sensitive biological component. The sugar forms an
amorphous solid matrix. However, the histone may have immunological
consequences if the preserved biological component is administered to a
human or animal.

SUMMARY OF THE INVENTION

[0005]The present inventor has found that viral preparations mixed with an
aqueous solution containing one, two or more sugars and a
polyethyleneimine (PEI) are preserved on drying such as on freeze-drying.
The addition of one or more sugars to a viral preparation leads to some
preservation of viral infectivity and/or immunogenicity. However, the
addition of PEI together with one or more sugars surprisingly leads to
improved preservation of viral infectivity and/or immunogenicity. A
particularly preferred improvement in infectivity and/or immunogenicity
is seen at relatively low concentrations of PEI and relatively high
concentrations of one or more sugars.

[0006]Accordingly, the present invention provides a method for preserving
viral particles comprising: [0007](i) providing an aqueous solution of
one or more sugars, a polyethyleneimine and said viral particles wherein
the concentration of polyethyleneimine is 15 μM or less based on the
number-average molar mass (Mn) of the polyethyleneimine and the
sugar concentration or, if more than one sugar is present, total sugar
concentration is greater than 0.1M; and [0008](ii) drying the solution to
form an amorphous solid matrix comprising said viral particles.

[0009]The invention further provides: [0010]a preserved product
comprising viral particles, one or more sugars and polyethyleneimine,
which product is in the form of an amorphous solid; [0011]an excipient
for the preservation of viral particles comprising: [0012](a) sucrose,
stachyose or raffinose or any combination thereof; and [0013](b)
polyethyleneimine at a concentration based on Mn of 5 μM or less;
[0014]use of the excipient for the preservation of viral particles during
and after freeze-drying. [0015]a kit comprising the excipient; [0016]a
vaccine comprising the preserved product and optionally an adjuvant;
[0017]a method of preparing a vaccine comprising viral particles, the
method comprising: [0018](a) providing an aqueous solution of one or more
sugars, a polyethyleneimine and said viral particles wherein the
concentration of polyethyleneimine is 15 μM or less based on the
number-average molar mass (Mn) of the polyethyleneimine and the
sugar concentration or, if more than one sugar is present, total sugar
concentration is greater than 0.1M; and [0019](b) optionally adding an
adjuvant, buffer, antibiotic and/or additive to the admixture; and
[0020](c) drying the solution to form an amorphous solid matrix
comprising said viral particles; and [0021]a dry powder comprising
preserved viral particles, obtainable by the method of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 shows the effect of an excipient composed of PEI, sucrose
(Suc) and raffinose (Raf) on Foot and Mouth Disease Virus (FMDV-A)
recovery following freeze-drying and either incubation for 24 hours at
room temperature (RT) or heat treatment for 48 at 37° C. (HT). The
results also show that phosphate buffered saline (PBS) offers no
protection during freeze-drying (FD). Error bars shown are standard error
of the mean (n=2).

[0023]FIG. 2 shows that PEI enhances the recovery of FMDV-O when added to
the sugars sucrose (Suc) and stachyose (Stac). The results obtained by
use of an excipient containing PEI, Suc and Stac are denoted by "Poly".
The results obtained by use of an excipient containing Suc and Stac
without PEI are denoted by "Sugar". The effect is seen with FMDV-O heat
treated for both 24 hours ("24 h HT") and 6 days ("6 day HT"). Error bars
shown are standard error of the mean (n=2).

[0024]FIG. 3 shows that the initial sugar concentration in the excipient
is important in maintaining FMDV-O stability. Diluting a solution of
sucrose and stachyose (120 Suc (3M):80 Stac (0.75M)) 1:10 by volume
produces a complete loss in the protective effects of a sugar only
excipient. Error bars shown are standard error of the mean (n=3).

[0025]FIG. 4.1 shows the effect of the sucrose (Suc):stachyose (Stac)
ratio on recovery of freeze-dried HT FMDV-O with varying PEI
concentrations. The results show that the addition of Stac increases
stability. The results for each PEI concentration were collated.
Statistical analysis using a one way ANOVA followed by a Turkey Test
showed that a mix containing all Suc achieved significantly lower
recovery than one containing a 140:60 by volume Suc:Stac ratio. Error
bars show standard error of the mean (n=8).

[0026]FIG. 4.2 shows the effect of Suc:Stac ratio on recovery of
freeze-dried HT FMDV-O with varying concentrations of histone (His)
according to WO-A-2006/0850082. The histone was histone 2A obtained from
Boehringer Mannheim. Error bars shown are standard error of the mean
(n=6).

[0027]FIG. 5 shows the effect of optimisation of the proportions of
Suc/Stac/PEI on adenovirus recovery. Plaque forming unit (PFU) formation
was assessed following freeze drying for 12 hours and heat treating for
24 hours. The results demonstrate when, using the highest levels of Stac,
virus PFU dropped off rapidly. With no Stac present, PFU was dramatically
reduced. When freeze dried with only PBS, there were no PFU. Lower
dilutions of PEI also appeared to enhance virus recovery.

[0028]FIG. 6 shows adenovirus recovery in PEI and sugar. PEI and sugar
excipient were optimised and compared to 3 controls; sugars only, PBS and
the original titre (before freeze drying). Freeze drying with PBS only
showed no thermoprotection. Sugars only showed some viral activity,
however, greater activity was seen using PEI together with sugar in the
excipient. Error bars shown are standard error of the mean (n=2).

[0029]FIG. 7 shows that an excipient containing Suc (1.5M)/Stac (0.125M)
with PEI at a concentration of 0.02 nM appears optimal and demonstrates
improved recovery of adenovirus over just a Suc Stac solution alone. PEI
as the sole excipient showed little to no protection during freeze
drying.

[0030]FIG. 8 shows the results of an experiment designed to compare two
different PEIs on adenoviral recovery. The high molecular weight PEI was
effective at a much lower concentration than the low molecular weight
PEI. PBS alone showed no virus recovery. Again optimal PEI/sugar
concentrations show improved recovery over sugars alone.

[0031]FIG. 9 show a comparison between diluting PEI in water ("PEIW") and
diluting PEI in PBS ("PEIP"). The results showed higher adenovirus titres
when PEI is diluted in PBS as opposed to water. When used as excipients
on their own both PBS and distilled water showed a very low level of
adenovirus recovery.

DETAILED DESCRIPTION OF THE INVENTION

Summary

[0032]The present invention relates to the preservation of viral particles
by contacting the viral particles with a preservation mixture. The
preservation mixture is an aqueous solution of PEI and one, two or more
sugars. Low concentrations of PEI and relatively high concentrations of
sugar are used. The solution in which the viral particles are present is
then dried to form an amorphous solid matrix comprising the viral
particles.

[0033]The invention thus enables virus structure and function to be
preserved during the drying step. Virus activity following drying can
thus be maintained. The preserved viral particles demonstrate improved
thermal and desiccation resistance allowing extension of shelf life, ease
of storage and transport and obviating the need for a cold chain for
distribution. The invention can thus provide protection as a
cryoprotectant (protection against freeze damage), lyoprotectant
(protection against desiccation) and/or a thermoprotectant (protection
against temperatures higher or lower than 4° C.).

[0035]The capsid is made up of subunits. Many viral capsids are made up of
subunits which may have either icosahedral or helical symmetry. Some
types of virus also include viral structures such as a nucleocapsid,
extra structures such as protein tails and complex outer walls, and
complex structures such as an icosahedral head bound to a helical tail.
The capsid may also be surrounded by an envelope. The envelope is
composed of lipoprotein bilayer and may contain material from the
membrane of a host cell as well as that of viral origin. For example, the
envelope may contain cell-derived lipids and virus-derived proteins.
Viruses may also contain glycoprotein spikes.

[0036]The viral particles used in the present invention may be whole
viruses such as live viruses, killed viruses, live attenuated viruses,
inactivated viruses such as chemically inactivated viruses or virulent or
non-virulent viruses. A live virus is capable of infecting and being
reproduced by the viral host. A killed virus is inactivated and is unable
to infect the viral host. The particles may be virus-like particles
(VLPs) or nucleocapsids.

[0037]The viral particle may be or may be derived from a dsDNA virus, a
ssDNA virus, a dsRNA virus, a (+)ssRNA virus, a (-)ssRNA virus, a
ssRNA-RT virus or a dsDNA-RT virus. As an example but not intended to be
limiting, the viral particle can be or can be derived from a virus of the
following families: [0038]adenoviridae such as human adenovirus A, B,
C, D, E or F including human Ad5, Ad2, Ad6, Ad24 serotypes;
[0039]caliciviridae such as the norwalk virus; [0040]coronaviridae such
as human coronavirus 299E or OC43 and SARS-coronavirus; [0041]filoviridae
such as ebola virus; [0042]flaviviridae such as yellow fever virus, west
nile virus, dengue virus, hepatitis C virus; [0043]hepadnaviridae such as
hepatitis B virus; [0044]herpesviridae such as herpes simplexvirus, human
herpesvirus 1, 3, 4, 5 or 6; [0045]orthomyxoviridae such as
influenzavirus A, B, C including but not limited to influenza A virus
serotypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9H2, H7N2, H7N3 and N10N7;
[0046]papillomaviridae such as human papilloma virus;
[0047]paramyxoviridae such as human parainfluenza virus 1, measles virus
and mumps virus; [0048]parvoviridae such as adeno-associated virus;
[0049]picornaviridae such as human poliovirus, foot and mouth disease
virus (including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1);
[0050]poxyiridae such as vaccinia virus, variola virus and avian poxvirus
(fowlpox); [0051]reoviridae such as bluetongue virus group;
[0052]retroviridae such as lentivirus including human immunodeficiency
virus 1 and 2; and [0053]togaviridae such as rubella virus.

[0054]In a preferred embodiment, the viral particle can be or can be
derived from an adenoviridae, orthomyxoviridae, parvoviridae,
picornaviridae or poxyiridae virus. In a particularly preferred
embodiment, the viral particle can be or can be derived from an
adenovirus, vaccinia virus, influenza virus, or foot and mouth disease
virus.

[0056]Viral particles can be prepared using standard techniques well known
to those skilled in the art. For example, a virus may be prepared by
infecting cultured host cells with the virus strain that is to be used,
allowing infection to progress such that the virus replicates in the
cultured cells and can be released by standard methods known in the art
for harvesting and purifying viruses.

Preservation Mixture

[0057]The preservation mixture of the present invention comprises an
aqueous solution of one or more sugars and a polyethyleneimine (PEI). Any
suitable aqueous solution may be used. The solution may be buffered. The
solution may be a HEPES solution, phosphate-buffered saline (PBS) or pure
water.

[0058]Sugars suitable for use in the present invention include reducing
sugars such as glucose, fructose, glyceraldehydes, lactose, arabinose and
maltose; and non-reducing sugars such as sucrose. The sugar may be a
monosaccharide, disaccharide, trisaccharide, or other oligosaccharides.
The term "sugar" includes sugar alcohols.

[0059]Monosaccharides such as galactose, raffinose and mannose;
dissaccharides such as lactose and maltose; and tetrasaccharides such as
stachyose are envisaged. Trehalose, umbelliferose, verbascose,
isomaltose, cellobiose, maltulose, turanose, melezitose and melibiose are
also suitable for use in the present invention. A suitable sugar alcohol
is mannitol.

[0060]Preferably, the aqueous solution of one, two or more sugars is a
solution of sucrose, raffinose or stachyose. In particular, sucrose is a
disaccharide of glucose and fructose; raffinose is a trisaccharide
composed of galactose, fructose and glucose; and stachyose is a
tetrasaccharide consisting of two Da-galactose units, one
Dα-glucose unit and one Dβ-fructose unit sequentially linked.
A combination of sucrose and raffinose, or of sucrose and stachyose may
be employed.

[0061]Preservation of viral infectivity or immunogenicity is particularly
effective when at least two sugars are used in the preservation mixture
of the present invention. Therefore, the solution of one or more sugars
comprises a solution of at least 2, at least 3, at least 4 or at least 5
sugars. Combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, etc sugars are
envisaged. Preferably, the solution of two or more sugars comprises
sucrose and raffinose, or sucrose and stachyose.

[0062]PEI is an aliphatic polyamine characterised by the repeating
chemical units denoted as --(CH2--CH2--NH)--. Reference to PEI
herein includes a polyethyleneimine homopolymer or copolymer. The
polyethyleneimine copolymer may be a random or block copolymer. For
example, PEI may consist of a copolymer of polyethyleneimine and another
polymer such as polyethylene glycol (PEG). The polyethyleneimine may be
linear or branched.

[0063]Reference to PEI also includes derivatised forms of a
polyethyleneimine. A polyethyleneimine contains nitrogen atoms at various
positions. Nitrogen atoms are present in terminal amino groups, e.g.
R--NH2, and in internal groups such as groups interrupting an alkyl
or alkylene group within the polymer structure, e.g. R--N(H)--R', and at
the intersection of a polymer branch, e.g. R--N(--R')-R'' wherein R, R'
and R'' may be alkylene groups for example. Alkyl or aryl groups may be
linked to the nitrogen centres in addition to or instead of hydrogen
atoms. Such alkyl and aryl groups may be substituted or unsubstituted. An
alkyl group would be typically a C1-C4 alkyl group, e.g.
methyl, ethyl, propyl, isopropyl, butyl, sec.butyl or tert.butyl. The
aryl group is typically phenyl.

[0064]The PEI may be a polyethyleneimine that has been covalently linked
to a variety of other polymers such as polyethylene glycol. Other
modified versions of PEI have been generated and some are available
commercially: branched PEI 25 kDa, jetPEI®, LMW-PEI 5.4 kDa,
Pseudodendrimeric PEI, PEI-SS-PEI, PEI-SS-PEG, PEI-g-PEG, PEG-co-PEI,
PEG-g-PEI, PEI-co-L lactamide-co-succinamide,
PEI-co-N-(2-hydroxyethyl-ethylene imine), PEI-co-N-(2-hydroxypropyl)
methacrylamide, PEI-g-PCL-block-PEG, PEI-SS-PHMPA, PEI-g-dextran 10 000
and PEI-g-transferrin-PEG, Pluronic85®/Pluronic123®-g-PEI.

[0065]PEI is available in a broad range of number-average molar masses
(Mn) for example between 300 Da and 800 kDa. Preferably, the
number-average molar mass is between 300 and 2000 Da, between 500 and
1500 Da, between 1000 and 1500 Da, between 10 and 100 kDa, between 20 and
100 kDa, between 30 and 100 kDa, between 40 and 100 kDa, between 50 and
100 kDa, between 60 and 100 kDa, between 50 and 70 kDa or between 55 and
65 kDa. A relatively high Mn PEI of approximately 60 kDa or a
relatively low Mn of 1200 Da is suitable.

[0066]Preferably, the weight-average molar mass (Mw) of PEI is
between 500 Da and 1000 kDa. Most preferably, the Mw of PEI is
between 500 Da and 2000 Da, between 1000 Da and 1500 Da, or between 1 and
1000 kDa, between 100 and 1000 kDa, between 250 and 1000 kDa, between 500
and 1000 kDa, between 600 and 1000 kDa, between 750 and 1000 kDa, between
600 and 800 kDa, between 700 and 800 kDa. A relatively high Mw of
approximately 750 kDa or a relatively low Mw of approximately 1300
Da is suitable.

[0067]The weight-average molar mass (Mw) and number-average molar
mass (Mn) of PEI can be determined by methods well known to those
skilled in the art. For example, Mw may be determined by light
scattering, small angle neutron scattering (SANS), X-ray scattering or
sedimentation velocity. Mn may be determined for example by gel
permeation chromatography, viscometry (Mark-Houwink equation) and
colligative methods such as vapour pressure osometry or end-group
titration.

[0068]Various forms of PEI are available commercially (e.g. Sigma,
Aldrich). For example, a branched, relatively high molecular weight form
of PEI used herein with an Mn of approximately 60 kDa and a Mw
of approximately 750 kDa is available commercially (Sigma P3143). This
PEI can be represented by the following formula:

##STR00001##

[0069]A relatively low molecular weight form of PEI used herein is also
available commercially (e.g. Aldrich 482595) which has a Mw of 1300
Da and Mn of 1200 Da.

[0070]In the present invention, a preservation mixture comprising an
aqueous solution of PEI and one, two or more sugars is provided.
Typically, the viral particles are admixed with the preservation mixture
to provide the aqueous solution for drying.

[0071]The concentration of sugar in the aqueous solution for drying is
greater than 0.1M. Preferably, the concentration of the sugar in the
aqueous solution for drying or, if more than one sugar is present, the
total concentration of sugar in the aqueous solution for drying, is at
least 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.75M, 0.9M, 1M or 2M up to
saturation e.g. saturation at room temperature or up to 3M, 2.5M or 2M.
The sugar concentration or the total concentration if more than one sugar
is present may be from 0.5 to 2M. When more than one sugar is present,
each sugar may be present at a concentration of from 0.2M, 0.3M, 0.4M,
0.5M, 0.6M, 0.75M, 0.9M, 1M or 2M up to saturation e.g. saturation at
room temperature or up to 3M, 2.5M or 2M.

[0072]The concentration of PEI in the aqueous solution for drying is
generally in the range of 15 μM or less based on Mn. The PEI
concentration may be 10 μM or less based on Mw. Such
concentrations of PEI are particularly effective at preserving viral
infectivity or immunogenicity.

[0073]In a preferred embodiment of the invention, the PEI is provided at a
concentration based on Mn of less than 5 μM, less than 500 nM,
less than 100 nM, less than 40 nM, less than 25 nM, less than 10 nM, less
than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than
0.1 nM, less than 0.075 nM, less than 0.05 nM, less than 0.025 Nm or less
than 0.0025 nM. Typically the PEI concentration based on Mn is
0.0025 nM or more, 0.025 nM or more, or 0.1 nM or more. A suitable PEI
concentration range based on Mn is between 0.0025 nM and 5 μM, or
between 0.025 and 200 nM.

[0074]Preferably, the PEI concentration based on Mw is less than 5
μM, less than 1 μM, less than 0.1 μM, less than 0.1 μM, less
than 5 nM, less than 4 nM, less than 2 nM, less than 1 nM, less than 0.5
nM, less than 0.25 nM, less than 0.1 nM, less than 0.05 nM, less than
0.02 nM, less than 0.002 nM or less than 0.1 nM. Typically the PEI
concentration based on Mw is 0.00001 nM or more, 0.001 nM or more or
0.01 nM or more. A suitable PEI concentration range based on Mw is
between 0.00001 and 20 nM, between 0.0001 and 20 nM or between 0.0001 and
5 nM.

[0075]Typically, it is found that relatively high molecular weight PEI is
effective at lower concentrations than relatively low molecular weight
PEI. Thus: [0076]Where a relatively high Mw PEI is used, for
example in the range of 20 to 1000 kDa, a concentration of PEI of between
0.001 and 5 nM based on Mw is preferred. Where a relatively low
Mw PEI is used, for example in the range of 300 Da to 10 kDa, a
concentration of PEI of between 0.0001 and 10 μM is preferred.
[0077]Where a relatively high Mn PEI is used, for example in the
range of 20 to 1000 kDa, the concentration of PEI based on Mn is
preferably between 0.001 and 100 nM. Where a relatively low Mn, is
used, for example in the range of 1 Da to 10 kDa, a concentration of PEI
of between 0.0001 and 10 μM is used.

[0078]In an embodiment, the preservation mixture initially contacted with
the viral particles comprises PEI at a concentration based on Mn of
less than 2 μM and a solution of one or more sugars at a concentration
of at least 0.1M, at least 0.2M, at least 0.3M, at least 0.4M, at least
0.5M, at least 0.75M, at least 0.9M, at least 1M, or at least 2M.

[0079]When the solution of one or more sugars comprises two or more
sugars, the most effective concentration of PEI will be dependent on the
particular type of sugar used in the preservation mixture. For example,
when one of the two or more sugars is sucrose and the other is stachyose,
PEI at a concentration based on Mn of less than 2 μM, in
particular at a concentration between 0.025 nM and 2 μM, is effective
at preservation. In a preferred embodiment, the method of the invention
involves admixing the viral particles with an aqueous solution of (i) one
or more sugars wherein one of these sugars is sucrose and the other is
stachyose and (ii) PEI at a concentration based on Mn of less than 2
μM.

[0080]When the aqueous solution of two or more sugars comprises an aqueous
solution of sucrose and raffinose the preferred concentration of PEI is
found to be less than 2 μM, or in the range between 0.0025 nM and 2
μM. Therefore in a further embodiment, the method of the invention
involves admixing the viral particles with an aqueous solution of (i)
sucrose and raffinose and (ii) PEI at a concentration between 0.0025 nM
and 2 μM. Preferably, when a relatively high molecular weight PEI is
used, for example between 10 and 100 kDa based on Mn, the
concentration of PEI based on Mn is between 0.1 and 100 nM.

[0081]Whilst using a combination of two sugars in the preservation
mixture, the present inventors investigated the effect of different molar
concentration ratios of these sugars on the preservation of the viral
particle. Specific molar concentration ratios of one sugar to another
were particularly effective but the exact ratio depended on the types of
sugar used. Therefore in one embodiment of the invention in which one of
the two or more sugars comprises sucrose, the concentration of sucrose
relative to the other sugar is at a ratio of molar concentrations of
between 3:7 and 9:1, preferably at a ratio of at least 4:6, at least
50:50, at least 6:4, at least 7:3, at least 8:2 or at least 9:1. In the
case of sucrose and stachyose, a ratio of molar concentrations of
sucrose:stachyose of at least 3:7, at least 4:6, at least 50:50, at least
6:4, at least 7:3, at least 3:1, at least 8:2 or at least 9:1
demonstrated particularly effective preservation. Preferably, the
solution of two or more sugars comprises a solution of sucrose and
stachyose at a ratio of molar concentrations of between 50:50 and 8:2.

[0082]In a further embodiment, the preservation mixture of the present
invention comprises an aqueous solution of (i) two or more sugars in
which one of the sugars is sucrose and the concentration of sucrose
relative to the other sugar is at a ratio of molar concentrations between
3:7 and 9:1 and (ii) PEI at a concentration of less than 100 nM or at a
concentration based on Mn between 0.025 and 100 nM.

Preservation

[0083]The preservation techniques of the present invention are
particularly suited to preservation against desiccation and freezing of
viral particles and thermal challenge. Preservation of viral particles is
achieved by drying viral particles admixed with the preservation mixture
of the present invention. On drying, an amorphous solid is formed. By
"amorphous" is meant non-structured and having no observable regular or
repeated organization of molecules (i.e. non-crystalline).

[0084]Typically, drying is achieved by freeze-drying, snap-freezing,
vacuum drying or spray-drying. Freeze-drying is preferred. By removing
the water from the material and sealing the material in a vial, the
material can be easily stored, shipped and later reconstituted to its
original form.

[0085]Freeze-drying is a dehydration process typically used to preserve
perishable material or make the material more convenient for transport.
Freeze-drying represents a key step for manufacturing solid protein and
vaccine pharmaceuticals. However, biological materials are subject to
both freezing and drying stresses during the procedure, which are capable
of unfolding or denaturing proteins. Furthermore, the rate of water
vapour diffusion from the frozen biological material is very low and
therefore the process is time-consuming. The preservation technique of
the present invention enables biological materials to be protected
against the desiccation and/or thermal stresses of the freeze-drying
procedure.

[0086]There are three main stages to this technique namely freezing,
primary drying and secondary drying. Freezing is typically performed
using a freeze-drying machine. In this step, it is important to cool the
biological material below its eutectic point, the lowest temperature at
which the solid and liquid phase of the material can coexist. This
ensures that sublimation rather than melting will occur in the following
steps. Alternatively, amorphous materials do not have a eutectic point,
but do have a critical point, below which the product must be maintained
to prevent melt-back or collapse during primary and secondary drying.

[0087]During primary drying the pressure is lowered and enough heat
supplied to the material for the water to sublimate. The amount of heat
necessary can be calculated using the sublimating molecules' latent heat
of sublimation. About 95% of the water in the material is sublimated at
this stage. Primary drying may be slow as too much heat could degrade or
alter the structure of the biological material. In order to control the
pressure, a partial vacuum is applied which speeds sublimation. A cold
condenser chamber and/or condenser plates provide a surface(s) for the
water vapour to re-solidify on.

[0088]In the secondary drying process, water molecules adsorbed during the
freezing process are sublimated. The temperature is raised higher than in
the primary drying phase to break any physico-chemical interactions that
have formed between the water molecules and the frozen biological
material. Typically, the pressure is also lowered to encourage
sublimation. After completion of the freeze-drying process, the vacuum is
usually broken with an inert gas, such as nitrogen, before the material
is sealed.

[0089]In one embodiment, drying is achieved by freezing the mixture, such
as by snap freezing. The term "snap freezing" means a virtually
instantaneous freezing as is achieved, for example, by immersing a
product in liquid nitrogen. In some embodiments it refers to a freezing
step, which takes less than 1 to 2 seconds to complete.

[0090]In certain embodiments, drying is carried out using vacuum
desiccation at around 1300 Pa. However vacuum desiccation is not
essential to the invention and in other embodiments, the preservation
mixture contacted with the viral particle is spun (i.e. rotary
desiccation) or freeze-dried (as further described below).
Advantageously, the method of the invention further comprises subjecting
the preservation mixture containing the viral particle to a vacuum.
Conveniently, the vacuum is applied at a pressure of 20,000 Pa or less,
preferably 10,000 Pa or less. Advantageously, the vacuum is applied for a
period of at least 10 hours, preferably 16 hours or more. As known to
those skilled in the art, the period of vacuum application will depend on
the size of the sample, the machinery used and other parameters.

[0091]In another embodiment, drying is achieved by spray-drying the viral
particles admixed with the preservation mixture of the invention. This
technique is well known to those skilled in the art and involves a method
of drying a liquid feed through a hot gas e.g. air, oxygen-free gas or
nitrogen. The liquid feed is atomized into a spray of droplets. The
droplets are then dried by contact with the hot gas in a drying chamber.

[0092]Once mixed with the preservation mixture, the samples can be dried
to various residual moisture contents to offer long term preservation at
greater than refrigeration temperatures e.g. within the range from about
4° C. to about 45° C., or lower than refrigeration
temperatures e.g. within the range from about 0 to -70° C. or
below.

[0093]Using the method of the invention, the admixture of viral particles
and preservation mixture is dried to form an amorphous solid matrix. In
one embodiment of the invention, the amorphous solid is obtained in a dry
powder form. The amorphous solid may take the form of free-flowing
particles.

[0094]However, in a further embodiment of the method of the invention, the
admixture comprising viral particles is dried onto a solid support. The
solid support may comprise a bead, test tube, matrix, plastic support,
microtiter dish, microchip (for example, silicon, silicon-glass or gold
chip), or membrane. In another embodiment, there is provided a solid
support onto which a viral particle preserved according to the methods of
the present invention is dried or attached.

[0095]The present invention provides a kit comprising an excipient
comprising an aqueous solution of (i) one or more sugars and (ii) PEI.
Preferably the kit comprises further excipients, carriers or diluents
suitable for veterinary or pharmaceutical purposes. Instructions for
administration may also be provided. The kit may also comprise auxiliary
substances, such as adjuvants, setting or emulsifying agents, pH buffer
agents, gelling or viscosity enhancing additives or colours, suitable for
delivery of the preserved viral particles of the invention into a patient
or target cell.

[0097]The preservation of viral particles in accordance with the present
invention can be measured by the assessment of parameters such as viral
infectivity, immunogenicity, transfection rates, virus titre and host
cell response. Such techniques are well known to those skilled in the
art.

[0098]Methods of assaying for viral infectivity and/or immunogenicity are
well known to those skilled in the art and include but are not limited to
growth of a virus in a cell culture, detection of virus-specific antibody
in blood, ability to elicit T and/or B cell responses, detection of viral
antigens, detection of virus encoded DNA or RNA, or observation of virus
particles using a microscope.

[0099]Further, the presence of a virus gives rise to morphological changes
in the host cell, which can be measured to give an indication of viral
activity. Detectable changes such as these in the host cell due to viral
infection are known as cytopathic effect. Cytopathic effects may consist
of cell rounding, disorientation, swelling or shrinking, death and
detachment from the surface. Many viruses induce apoptosis (programmed
cell death) in infected cells, measurable by techniques such as the TUNEL
(Terminal uridine deoxynucleotidyl transferase dUTP nick end labelling)
assay and other techniques well known to those skilled in the art.

[0100]Viruses may also affect the regulation of expression of the host
cell genes and these genes can be analysed to give an indication of
whether viral activity is present or not. Such techniques may involve the
addition of reagents to the cell culture to complete an enzymatic or
chemical reaction with a viral expression product. Furthermore, the viral
genome may be modified in order to enhance detection of viral
infectivity. For example, the viral genome may be genetically modified to
express a marker that can be readily detected by phase contrast
microscopy, fluorescence microscopy or by radioimaging. The marker may be
an expressed fluorescent protein such as GFP (Green Fluorescent Protein)
or an expressed enzyme that may be involved in a colourimetric or
radiolabelling reaction. The marker could also be a gene product that
interrupts or inhibits a particular function of the cells being tested.

[0101]An assay for plaque-forming units can be used to measure viral
infectivity and to indicate viral titre. In this assay, suitable host
cells are grown on a flat surface until they form a monolayer of cells
covering a plastic bottle or dish. The selection of a particular host
cell will depend on the type of virus. Examples of suitable host cells
include but are not limited to CHO, BHK, MDCK, 10T1/2, WEHI cells, COS,
BSC 1, BSC 40, BMT 10, VERO, W138, MRC5, A549, HT1080, 293, B-50, 3T3,
NIH3T3, HepG2, Saos-2, Huh7, HEK293 and HeLa cells. The monolayer of host
cells is then infected with the viral particles. The liquid medium is
replaced with a semi-solid one so that any virus particles produced, as
the result of an infection cannot move far from the site of their
production. A plaque is produced when a virus particle infects a cell,
replicates, and then kills that cell. A plaque refers to an area of cells
in the monolayer which display a cytopathic effect, e.g. appearing round
and darker than other cells under the microscope, or as white spots when
visualized by eye; the plaque center may lack cells due to virus-induced
lysis. The newly replicated virus infects surrounding cells and they too
are killed. This process may be repeated several times. The cells are
then stained with a dye such as methylene blue, which stains only living
cells. The dead cells in the plaque do not stain and appear as unstained
areas on a coloured background.

[0102]Each plaque is the result of infection of one cell by one virus
followed by replication and spreading of that virus. However, viruses
that do not kill cells may not produce plaques. A plaque refers to an
area of cells in a monolayer which display a cytopathic effect, e.g.
appearing round and darker than other cells under the microscope, or as
white spots when visualized by eye; the plaque center may lack cells due
to virus-induced lysis. An indication of viral titre is given by
measuring "plaque-forming units" (PFU). PFU refers to a virus or group of
viruses, which cause a plaque. For example: if a viral stock solution has
1000 pfu/ml, it means that every ml of this stock has 1000 virus
particles which can form plaques. Levels of viral infectivity can be
measured in a sample of biological material preserved according to the
present invention and compared to control samples such as freshly
harvested virus or samples subjected to desiccation and/or thermal
variation without addition of the preservation mixture of the present
invention.

[0103]Typically, the viral titre following preservation according to the
present invention and incubation of the resulting product at 37°
C. for 5 days is at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of
the titre of the virus prior to such incubation or, indeed prior to
preservation according to the present invention and such incubation.

[0104]Some types of viral particles of the invention, such as viral
proteins, VLPs, or some inactivated viruses do not have the ability to
form plaques in the plaque assay. In this case, preservation can be
measured by other methods such as methods for determining immunogenicity
which are well known to those skilled in the art. For example, in vivo
and in vitro assays for measuring antibody or cell-mediated host immune
responses are known in the art and suitable for use in the present
invention. For example, an antibody based immune response may be measured
by comparing the amount, avidity and isotype distribution of serum
antibodies in an animal model, before and after immunization using the
preserved viral particle of the invention.

Uses of the Preserved Viral Particles of the Invention

Vaccines

[0105]The preserved viral particles of the present invention may find use
as a vaccine. For example, preserved viral particles such as whole killed
virus, live attenuated virus, chemically inactivated virus, VLPs or live
viral vectors are suitable for use as a vaccine. As a vaccine the
preserved viral particles of the invention may be used as antigens or to
encode antigens such as viral proteins for the treatment or prevention of
a number of conditions including but not limited to viral infection,
sequelae of viral infection including but not limited to viral-induced
toxicity, cancer and allergies. Such antigens contain one or more
epitopes that will stimulate a host's immune system to generate a humoral
and/or cellular antigen-specific response.

[0107]The vaccine compositions of the present invention comprise viral
particles admixed with the preservation mixture of the invention
containing one or more sugars and PEI. The vaccine composition may
further comprise appropriate buffers and additives such as antibiotics,
adjuvants or other molecules that enhance presentation of vaccine
antigens to specific cells of the immune system.

[0108]A variety of adjuvants well known in the art can be used in order to
increase potency of the vaccine and/or modulate humoral and cellular
immune responses. Suitable adjuvants include, but are not limited to,
oil-in-water emulsion-containing adjuvants or water in oil adjuvants,
such as mineral oil, aluminium based adjuvants, squalene/phosphate based
adjuvants, Complete/Incomplete Freunds Adjuvant, cytokines and any other
substances that act as immunostimulating agents to enhance the
effectiveness of the vaccine.

[0109]The vaccine composition of the present invention can be in a
freeze-dried (lyophilised) form in order to provide for appropriate
storage and maximize the shelf-life of the preparation. This will allow
for stock piling of vaccine for prolonged periods of time and help
maintain immunogenicity, potency and efficacy. The preservation mixture
of the present invention is particularly suited to preserve viral
substances against desiccation and thermal stresses encountered during
freeze-drying/lyophilisation protocols. Therefore, the preservation
mixture is suitable for adding to the virus or viral particle soon after
harvesting and before subjection of the sample to the freeze-drying
procedure.

[0110]To measure the preservation of a vaccine prepared in accordance with
the present invention, the potency of the vaccine can be measured using
techniques well known to those skilled in the art. For example, the
generation of a cellular or humoral immune response can be tested in an
appropriate animal model by monitoring the generation of antibodies or
immune cell responses to the vaccine. The ability of vaccine samples
prepared in accordance with the method of the present invention to
trigger an immune response may be compared with vaccines not subjected to
the same preservation technique.

Viral Vectors

[0111]A virus or viral vector preserved according to the method of the
present invention can be used to transfer a heterologous gene or other
nucleic acid sequence to target cells. Suitably, the heterologous
sequence (i.e. transgene) encodes a protein or gene product which is
capable of being expressed in the target cell. Suitable transgenes
include desirable reporter genes, therapeutic genes and genes encoding
immunogenic polypeptides (for use as vaccines). Gene therapy, an approach
for treatment or prevention of diseases associated with defective gene
expression, involves the insertion of a therapeutic gene into cells,
followed by expression and production of the required proteins. This
approach enables replacement of damaged genes or inhibition of expression
of undesired genes. In particular, the preserved virus or viral vector
may be used in gene therapy to transfer a therapeutic transgene or gene
encoding immunogenic polypeptides to a patient.

[0112]In a preferred embodiment, the preserved viral particle is a live
viral vector. By "live viral vector" is meant a live viral vector that is
non-pathogenic or of low pathogenicity for the target species and in
which has been inserted one or more genes encoding antigens that
stimulate an immune response protective against other viruses or
microorganisms, a reporter gene or a therapeutic protein. In particular,
nucleic acid is introduced into the viral vector in such a way that it is
still able to replicate thereby expressing a polypeptide encoded by the
inserted nucleic acid sequence and in the case of a vaccine, eliciting an
immune response in the infected host animal. In one embodiment, the live
viral vector is an attenuated live viral vector i.e. is modified to be
less virulent (disease-causing) than wildtype virus.

[0113]The basis of using recombinant viruses as potential vaccines
involves the incorporation of specific genes from a pathogenic organism
into the genome of a nonpathogenic or attenuated virus. The recombinant
virus can then infect specific eukaryotic cells either in vivo or in
vitro, and cause them to express the recombinant protein.

[0114]Live viral vector vaccines derived by the insertion of genes
encoding sequences from disease organisms may be preferred over live
attenuated vaccines, inactivated vaccines, subunit or DNA approaches. One
of the most important safety features of live viral vectors is that the
recipients may be immunized against specific antigens from pathogenic
organisms without exposure to the disease agent itself. Safety is further
regulated by the selection of a viral vector that is either attenuated
for the host or unable to replicate in the host although still able to
express the heterologous antigen of interest. A vaccine strain that has a
history of safety in the target species offers an additional safety
feature. Several systems have been developed in which the vector is
deleted of essential genes and preparation of the vaccine is carried out
in cell systems that provide the missing function.

[0115]A variety of vectors such as retroviral, lentiviral, herpes virus,
poxvirus, adenoviral and adeno-associated viral vectors can be used for
the delivery of heterologous genes to target cells. The heterologous gene
of interest may be inserted into the viral vector. The viral vectors of
the invention may comprise for example a virus vector provided with an
origin of replication, optionally a promoter for the expression of the
heterologous gene and optionally a regulator of the promoter. For
example, adenoviruses useful in the practice of the present invention can
have deletions in the E1 and/or E3 and/or E4 region, or can otherwise be
maximized for receiving heterologous DNA.

[0116]The viral vector may comprise a constitutive promoter such as a
cytomegalovirus (CMV) promoter, SV40 large T antigen promoter, mouse
mammary tumour virus LTR promoter, adenovirus major late promoter (MLP),
the mouse mammary tumour virus LTR promoter, the SV40 early promoter,
adenovirus promoters such as the adenovirus major late promoter (Ad MLP),
HSV promoters (such as the HSV IE promoters), HPV promoters such as the
HPV upstream regulatory region (URR) or rous sarcoma virus promoter
together with other viral nucleic acid sequences operably linked to the
heterologous gene of interest. Tissue-specific or inducible promoters can
also be used to control expression of the heterologous gene of interest.
Promoters may also be selected to be compatible with the host cell for
which expression is designed.

[0117]The viral vector may also comprise other transcriptional modulator
elements such as enhancers. Enhancers are broadly defined as a cis-acting
agent, which when operably linked to a promoter/gene sequence, will
increase transcription of that gene sequence. Enhancers can function from
positions that are much further away from a sequence of interest than
other expression control elements (e.g. promoters) and may operate when
positioned in either orientation relative to the sequence of interest.
Enhancers have been identified from a number of viral sources, including
polyoma virus, BK virus, cytomegalovirus (CMV), adenovirus, simian virus
40 (SV40), Moloney sarcoma virus, bovine papilloma virus and Rous sarcoma
virus. Examples of suitable enhancers include the SV40 early gene
enhancer, the enhancer/promoter derived from the long terminal repeat
(LTR) of the Rous Sarcoma Virus, and elements derived from human or
murine CMV, for example, elements included in the CMV intron A sequence.

[0118]The viral vector containing a heterologous gene of interest may then
be preserved according to the method of the invention before storage,
subjecting to further preservation techniques such as lyophilisation, or
administration to a patient or host cell.

[0119]Nucleic acids encoding for polypeptides known to display antiviral
activity, immunomodulatory molecules such as cytokines (e.g. TNF-alpha,
interferons such as IL-6, and IL-2, interferons, colony stimulating
factors such as GM-CSF), adjuvants and co-stimulatory and accessory
molecules may be included in the viral vector of the invention.
Alternatively, such polypeptides may be provided separately, for example
in the preservation mixture of the invention or may be administrated
simultaneously, sequentially or separately with viral vectors of the
invention.

[0120]Preferably, the preserved viral vector of the invention may be
introduced into suitable host cells using a variety of viral techniques
that are known in the art, such as for example infection with recombinant
viral vectors such as retroviruses, herpes simplex virus and
adenoviruses. Preferably, administration of the preserved viral vector of
the invention containing a gene of interest is mediated by viral
infection of a target cell.

[0121]A number of viral based systems have been developed for transfecting
mammalian cells.

[0122]For example, a selected recombinant nucleic acid molecule can be
inserted into a vector and packaged as retroviral particles using
techniques known in the art. The recombinant virus can then be isolated
and delivered to cells of the subject either in vivo or ex vivo.
Retroviral vectors may be based upon the Moloney murine leukaemia virus
(Mo-MLV). In a retroviral vector, one or more of the viral genes (gag,
pol & env) are generally replaced with the gene of interest.

[0123]A number of adenovirus vectors are known. Adenovirus subgroup C
serotypes 2 and 5 are commonly used as vectors. The wild type adenovirus
genome is approximately 35 kb of which up to 30 kb can be replaced with
foreign DNA.

[0124]There are four early transcriptional units (E1, E2, E3 & E4), which
have regulatory functions, and a late transcript, which codes for
structural proteins. Adenovirus vectors may have the E1 and/or E3 gene
inactivated. The missing gene(s) may then be supplied in trans either by
a helper virus, plasmid or integrated into a helper cell genome.
Adenovirus vectors may use an E2a temperature sensitive mutant or an E4
deletion. Minimal adenovirus vectors may contain only the inverted
terminal repeats (ITRs) & a packaging sequence around the transgene, all
the necessary viral genes being provided in trans by a helper virus.
Suitable adenoviral vectors thus include Ad5 vectors and simian
adenovirus vectors.

[0125]Viral vectors may also be derived from the pox family of viruses,
including vaccinia viruses and avian poxvirus such as fowlpox vaccines.
For example, modified vaccinia virus Ankara (MVA) is a strain of vaccinia
virus which does not replicate in most cell types, including normal human
tissues. A recombinant MVA vector may therefore be used to deliver the
polypeptide of the invention.

[0126]Addition types of virus such as adeno-associated virus (AAV) and
herpes simplex virus (HSV) may also be used to develop suitable vector
systems

Excipient

[0127]In the present invention, an excipient for the preservation of viral
particles is also provided. The excipient comprises (a) sucrose,
stachyose, trehalose, a sugar alcohol or raffinose or any combination
thereof; and (b) PEI at a concentration based on Mn of 5M or less.
By "excipient" is meant an inactive substance used as a carrier for the
viral particles of the invention (for example when the viral particles
are used as a vaccine). Typically, the viral particles (e.g. for use as a
vaccine) are dissolved into or mixed with the excipient, which acts as a
preservative of the viral particle and/or in some contexts aids
administration and absorption into the body. As well as the preservation
mixture of the present invention, an excipient may also comprise other
preservatives such as antioxidants, lubricants and binders well known in
the art, as long as those ingredients do not significantly reduce the
effectiveness of the preservation mixture of the present invention.

Assaying on a Solid Support

[0128]Preserved viral particles stored on a solid support may be used for
diagnostic purposes or to monitor a vaccination regime. For example, a
patient sample such as bodily fluid (blood, urine, saliva, phlegm,
gastric juices etc) may be preserved according to the methods described
herein by drying an admixture comprising the patient sample and
preservation mixture of the present invention onto a solid support.
Preserved patient samples may then be tested for the presence of viral
antigens/epitopes in the sample using anti-viral antibodies (for example
using ELISA). Alternatively, viral particles of interest may be preserved
according to the methods described herein by drying an admixture
comprising the viral particles and preservation mixture of the present
invention onto a solid support. Patient samples may be tested for the
presence of anti-viral antibodies by contacting the patient sample with a
solid support onto which the viral particles of interest are attached.
The formation of antigen-antibody complexes can elicit a measurable
signal. The presence and/or amount of viral particle antigen-antibody
complexes in a sample may be used to indicate the presence of a viral
infection or progress of a vaccination regime in a patient.

Administration

[0129]Preserved vaccines or viral particles according to the present
invention may be administered, in some instances after reconstitution of
a freeze-dried product, to a subject in vivo using a variety of known
routes and techniques. For example, the preserved vaccines can be
provided as an injectable solution, suspension or emulsion and
administered via parenteral, subcutaneous, oral, epidermal, intradermal,
intramuscular, interarterial, intraperitoneal, intravenous injection
using a conventional needle and syringe, or using a liquid jet injection
system. Preserved vaccines may be administered topically to skin or
mucosal tissue, such as nasally, intratrachealy, intestinal,
sublingually, rectally or vaginally, or provided as a finely divided
spray suitable for respiratory or pulmonary administration.

[0130]In one embodiment, the method of the invention further comprises the
step of processing the mixture into a formulation suitable for
administration as a liquid injection. Preferably, the method further
comprises the step of processing the mixture into a formulation suitable
for administration via ingestion or via the pulmonary route.

[0131]The preserved product is administered to a subject in an amount that
is compatible with the dosage formulation and that will be
prophylactically and/or therapeutically effective. The administration of
the preserved product or vaccine of the invention may be for either
"prophylactic" or "therapeutic" purpose. As used herein, the term
"therapeutic" or "treatment" includes any of the following: the
prevention of infection or reinfection; the reduction or elimination of
symptoms; and the reduction or complete elimination of a pathogen.
Treatment may be effected prophylactically (prior to infection) or
therapeutically (following infection).

[0132]The following Examples illustrate the invention. The PEI used in
Example 1, 2, 3, 4, 5, 6, 7, 8 and 10 had an Mw of 750000 and an
Mn of 60000 (Sigma P3143). The "high molecular weight" PEI used in
Example 9 had a Mw of 750000 and an Mn of 60000 (Sigma P3143).
The "low molecular weight" PEI of Example 9 had a Mw of 1300 and an
Mn of 1200 (Aldrich 482595). The histone used in the Examples was
histone 2A obtained from Boehringer Mannheim.

[0133]The following general experimental techniques were employed:

Freeze Drying

[0134]After vortexing, vials were frozen at -80° C. in freeze dryer
trays containing 30 ml water with rubber stoppers partially in. Frozen
vials were transferred to the freeze dryer stoppering shelf (Thermo
Fisher) of the pre-cooled freeze dryer (Thermo Fisher) and dried for 16
hours. Rubber stoppers were lowered fully into the vials under a vacuum
before removing from freeze dryer.

[0136]BHK-21 cells were prepared in 6-well tissue culture plates and grown
overnight at 37° C. until approximately 80-90% confluent.
Freeze-dried samples were re-suspended in DMEM. Cell monolayers were
washed with Phosphate Buffered Saline (PBS) and incubated with 100 μl
of FMDV sample at 37° C. for 15 minutes. Cell monolayers were
overlaid with 2 ml of Eagles overlay which was allowed to set at room
temperature (RT) before incubating at 37° C. for a further 40-48
hours.

[0138]96 flat bottomed cell culture dishes (Jencons, UK) were seeded with
HEK 293 cells at 105 cells per ml (100 μl per well) and
maintained at 37° C. with 5% CO2. After achieving 90%
confluence vials containing the adenovirus plus excipient were
reconstituted in 1 ml of DMEM plus 5% FBS. A 1:10 dilution step was then
taken by taking 100 ml from the reconstituted vial and adding to 900 ml
of DMEM. 100 ml of the resulting diluted virus was then added to the
first row on the plate and a 1:2 dilution carried out down the plate. The
process was then repeated with the next excipient. After a further 48
hours, the number GFP cells per well were counted using fluorescent
microscopy.

Statistical Analysis

[0139]A student T-test was performed to analyse significance between
different excipients using PRISM Graphpad software version 4.00.
Alternatively, where multiple comparisons of pairs were necessary, a one
way ANOVA was carried out with as Turkey post comparison test. The P
value summaries are *p<0.10; **p<0.05; and ***p<0.005.

Example 1

[0140]A recombinant adenovirus expressing enhanced green fluorescent
protein (EGFP) with a titre of 4.1×107 pfu/ml (per ml tissue
culture medium) was mixed (1:5 v/v) with an excipient comprising sucrose
(a saturated solution, approximately 64% w/v), stachyose (a saturated
solution, approximately 64% w/v) and PEI (33 μg/ml) at a ratio of
3:1:1 v/v respectively. The mixture was freeze-dried as follows: samples
were frozen in liquid nitrogen, and dried under vacuum at room
temperature for 16 hours. After this time samples were stored until use
at -20° C. or used immediately. Adenovirus was assayed using a
plaque assay in 293A cells. The results are shown in the following Table.

[0141]This experiment was designed to examine the effect of excipient
components on recovery of FMDV-A when freeze-dried (FD) and left for 24
hours at room temperature (RT) or heat treated for 48 hours at 37°
C. (HT). All excipients were prepared in glass vials. All vials were set
up in duplicate.

[0142]170 μl of an aqueous solution of Suc (1 g/ml) and 30 μl of an
aqueous solution of Raf (1 g/ml) were added to each other, giving a total
200 μl volume for the sugar mix. 50 μl of PEI (0.03 mg/ml) was then
added to complete the excipient. Finally, 50 μl of FMDV-A were added
and the mixture vortexed. The final concentration of each sugar and of
PEI in the excipient mixture is shown in the Table below:

[0143]A control mixture was prepared by addition of 50 μl of FMDV-A to
250 μl of PBS. The vials were freeze-dried and then left at RT for 24
hours or 37° C. for 48 hours. An FMDV assay was then performed.
The results are shown in FIG. 1. The results demonstrate that there is
very little virus recovery when the excipient was PBS only. The excipient
containing Suc, Raf and PEI demonstrated similar FMDV recovery at RT or
HT. A student T-test in fact showed no significant difference between
incubation at room temperature (RT) and heat treating for 48 hours at
37° C. (HT).

Example 3

[0144]This experiment was designed to investigate the benefit of PEI on
heat treated FMDV-O virus. Glass vials were prepared in duplicate with
120 μl Suc (3M), 80 μl Stac (0.75M) and 50 μl of either PEI
(10-2 mg/ml) or distilled water. 50 μl FMDV-O was added to each
excipient vial. The final concentration of each sugar and of PEI, when
present, in the excipient mixture is shown in the Table below:

[0145]50 μl Volumes of the virus used were also refrozen as controls
(original titre). After freeze-drying, samples were incubated at
37° C. for either 24 hours or 6 days. Samples were re-suspended in
1 ml DMEM (plus 10% FBS) and virus recovery determined in the plaque
assay. The results are shown in FIG. 2.

[0146]Example 3 was designed to assess the extra benefit of including PEI
in the sugar excipient over simply having the sugar excipient alone.
Following heat treatment at 37° C. for 24 hours, there was no
noticeable drop in virus recovery when the excipient containing PEI was
used whereas the recovery of virus was significantly reduced when the
sugar excipient was employed without PEI. After 6 days of heat treatment,
there was a loss of virus when the excipient containing PEI was used.
However, again, this loss was significantly lower than the loss when the
excipient containing sugar alone was employed.

Example 4

[0147]Initial sugar concentrations were examined to optimise the excipient
sugar component in the recovery of FMDV-O. A sugar solution with a 120:80
Suc (3M) and Stac (0.75M) ratio was prepared ("sugars") and 1:10, 1:100,
1:1000 serial dilutions carried out. Triplicate glass vials with 200
μl of each sugar concentration or PBS were prepared and 50 μl
FMDV-O added to the sugar solution or PBS.

[0148]The final concentration of each sugar is shown in the Table below:

[0149]50 μl volumes of the virus were also refrozen as controls
(original titre). Samples were freeze-dried, then incubated at 37°
C. for 7 days before re-suspension in 1 ml DMEM (plus 10% FBS) and virus
recovery determined in the plaque assay. The results are shown in FIG. 3.

[0150]The aim of Example 4 was to see the effect of diluting sugar
concentration. Following freeze drying, virus recovery drops to
approximately one tenth of the original titre in an excipient containing
the sugar solution. When the sugar concentration is diluted 1:10, no
significant recovery is seen.

Example 5

[0151]Example 5 was designed to investigate the effect of optimal
combinations of sucrose and stachyose concentrations for recovery of
FMDV-O. A series of sugar ratios from 80 μl Suc (3M) and 120 μl
Stac (0.75M) to 200 μl Suc and 1 μl Stac were prepared. PEI was
prepared with a series of dilutions of 1 mg/ml from 1:100 to 1:32000. His
was prepared with a series of dilutions from 1 mg/ml to 0.625 mg/ml. A
matrix of samples was prepared with 50 μl FMDV-O, 200 μl of each
sugar ratio and 50 μl each PEI dilution.

[0152]The final concentration of each sugar and of PEI is shown in the
Table below:

[0153]Samples were freeze-dried and incubated at 37° C. for 3 days
(PEI samples) or 24 hours (His samples) before re-suspending in 1 ml DMEM
plus 10% FBS) and determining FMDV recovery in the plaque assay. The
results are shown in FIGS. 4.1 and 4.2.

[0154]The results for each PEI concentration were collated. The results
shown in FIG. 4.1 demonstrate that a significantly lower recovery was
seen when using a sucrose only excipient compared to an excipient
containing a 140:60 ratio of Suc:Stac. Apart from a pure Suc excipient
there was no significant difference between the different ratios of
Suc:Stac.

[0155]FIG. 4.2 shows the results of the same experiment in which His is
replaced with PEI in the excipient. The results demonstrated an effect on
optimal Suc:Stac ratios with far higher ratios of Suc being preferable
and no noticeable deleterious of a Suc only excipient.

Example 6

[0156]In this Example, adenovirus with a GFP tag was used to compare virus
recovery with different sugar/PEI concentrations. A series of different
Suc:Stac ratios were set up with a final volume of 200 μl. 50 μl of
PEI at a range of concentrations from 0.1 mg per ml to 0.01 μg per ml
were also added to each vial. After addition of adenovirus to the
different Suc:Stac:PEI ratios, vials were frozen and FD.

[0157]The final concentration of each sugar and of PEI is shown in the
Table below:

[0158]Following 24 hours of heat treatment at 37° C., excipient and
virus were reconstituted in 1 ml of DMEM (plus 10% FBS). Virus titre was
assayed using 2 fold serial dilutions in 96 well plates containing a 90%
confluent monolayer of 293 cells. The results are shown in FIG. 5.

[0159]Initial optimisation examined three variables: Suc concentration,
Stac concentration and PEI concentration using a matrix of different
concentrations. The results of the matrix demonstrate that both sugar
concentration and PEI concentration can pronouncedly effect viral
recovery. High concentrations of Stac to Suc or high concentrations of
PEI both showed low levels of recovery. Optimal recovery of virus
required Suc to be present. Samples containing no Sue showed no recovery.

Example 7

[0160]The aim of this experiment was to examine PEI concentration in a
Suc:Stac sugar excipient. A series of 1:10 dilutions of PEI were set up
to assess optimal PEI concentrations. From the work on optimising sugar
concentrations, a ratio of 120:80 Suc:Stac was chosen.

[0161]The final concentration of each sugar and of PEI is shown in the
Table below:

[0162]Following freeze drying, samples were heat treated at 37° C.
for 24 hours then the adenovirus assay was carried out. The results are
shown in FIG. 6.

[0163]The results demonstrate that when PEI is used at the higher
concentrations low virus recovery is seen. As PEI is further diluted,
virus recovery improves with an optimal concentration of around 0.01
μg/ml. The recovery at this concentration was significantly higher
than virus recovery using sugar excipients only. Excipient containing PBS
showed no virus recovery.

Example 8

[0164]This experiment was designed to gain a greater understanding of the
different excipient components on virus stability during freeze drying.

[0165]The final concentration of each sugar and of PEI is shown in the
Table below:

[0166]Vials containing excipient plus virus were freeze dried over night.
Following freeze drying, vials were incubated at 37° C. for 5 days
prior to re-suspending in 1 ml of DMEM (10% FBS). The resulting solution
was then diluted 1:1000 before carrying out a series of 1:2 dilutions
prior to proceeding to the 293 assay. The results are shown in FIG. 7.

Example 9

[0167]Example 9 was designed to study the differences between high and low
molecular weight PEI. The high molecular weight PEI ("HPEI") has a
Mw of 750000 whereas the low molecular weight PEI ("LPEI") has a
Mw of 1300.

[0168]The final concentration of each sugar and of PEI is shown in the
Table below:

[0169]Vials containing excipient plus adenovirus were freeze dried over
night. Following freeze drying, vials were incubated at 37° C. for
5 days prior to re-suspending in 1 ml of DMEM (10% FBS). The resulting
solution was then diluted 1:1000 before carrying out a series of 1:2
dilutions prior to proceeding to the 293 assay. The results are shown in
FIG. 8.

Example 10

[0170]Example 10 was designed to examine the differences in adenovirus
recovery when PEI (high molecular weight; Mw 750000) was diluted in
PBS ("PEIP") or water ("PEIW").

[0171]The final concentration of each sugar and of PEI is shown in the
Table below:

[0172]Vials containing excipient plus virus were freeze dried over night.
Following freeze drying, vials were incubated at 37° C. for 5 days
prior to re-suspending in 1 ml of DMEM (10% FBS). The resulting solution
was then diluted 1:1000 before carrying out a series of 1:2 dilutions
prior to proceeding to the 293 assay. The results are shown in FIG. 9.